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Germline-specific dgcr8 knockout in zebrafish using a BACK approach

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Abstract

Zebrafish is an important model to study developmental biology and human diseases. However, an effective approach to achieve spatial and temporal gene knockout in zebrafish has not been well established. In this study, we have developed a new approach, namely bacterial artificial chromosome-rescue-based knockout (BACK), to achieve conditional gene knockout in zebrafish using the Cre/loxP system. We have successfully deleted the DiGeorge syndrome critical region gene 8 (dgcr8) in zebrafish germ line and demonstrated that the maternal-zygotic dgcr8 (MZdgcr8) embryos exhibit MZdicer-like phenotypes with morphological defects which could be rescued by miR-430, indicating that canonical microRNAs play critical role in early development. Our findings establish that Cre/loxP-mediated tissue-specific gene knockout could be achieved using this BACK strategy and that canonical microRNAs play important roles in early embryonic development in zebrafish.

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Acknowledgements

Special thanks are given to Dr. Koichi Kawakami (National Institute of Genetics, Japan) and Dr. Maximiliano L Suster (Uni Research AS, Norway) for the piTol2 plasmids. We thank Dr. Daiguan Yu (Guangzhou Institutes of Biomedicine and Health, China) for suggestions on the design of BAC recombineering, and Dr. Didier Stainier (Max Planck Institute for Heart and Lung Research, Germany) for critical comments on the manuscript. We thank Ms. Kathy W.Y. Sham for technical assistance and the Core Laboratories in the School of Biomedical Sciences for the provision of equipment and technical support.

This work was supported by the Research Grant Council of Hong Kong (Grant No. 14119715) and the Fundamental Research Funds for the Central Universities (Grant Nos. 151gzs102 and 151gzs121).

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Authors and Affiliations

  1. State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals, and the Guangdong Province Key Laboratory for Aquatic Economic Animals, Sun Yat-Sen University, Guangzhou, China

    Yun Liu & Yong Zhang

  2. School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China

    Yun Liu, Zeyao Zhu, Idy H. T. Ho, Yujian Shi, Yuxin Xie, Jianzhen Li, Yong Zhang & Christopher H. K. Cheng

  3. Department of Anaesthesia and Intensive Care, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China

    Idy H. T. Ho & Matthew T. V. Chan

  4. School of Biomedical Sciences Core Laboratory, The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, China

    Zeyao Zhu, Idy H. T. Ho, Yujian Shi, Yuxin Xie, Jianzhen Li & Christopher H. K. Cheng

  5. South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou, China

    Yong Zhang

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  1. Yun Liu
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Correspondence to Christopher H. K. Cheng.

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Y. Liu and Z. Zhu contributed equally to this work.

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Liu, Y., Zhu, Z., Ho, I.H.T. et al. Germline-specific dgcr8 knockout in zebrafish using a BACK approach. Cell. Mol. Life Sci. 74, 2503–2511 (2017). https://doi.org/10.1007/s00018-017-2471-7

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  • DOI: https://doi.org/10.1007/s00018-017-2471-7

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